A vaccine to combat malaria is a highly desirable public health tool to reduce morbidity and mortality in African children. In order to achieve this goal it will be essential to gain a detailed understanding of the impact of malaria on the generation and maintenance of immunological memory, the basis of all vaccines. Our goal is to gain an understanding of the generation, maintenance and activation of immunological memory in response to natural malaria infection, focusing on B cells and the function of Pf-specific antibodies. We determined the acquisition of both Pf-specific memory B cells (MBCs) and Pf-specific Abs using a proteome chip containing approximately 25% of the Pf proteome in a cohort of children and young adults in Kambila, Mali exposed to intense, seasonal Pf transmission. We documented that both the long-lived Pf-specific Abs and MBCs increased slowly, year by year, in a step-wise fashion. This year we established a collaboration with Drs. Turner and Lavstsen (U. Copenhagen) and Dr. Smith (Seattle Biomedical Research Institute) to characterize the acquisition of antibodies specific for the Pf variant antigen, Pf erythrocyte membrane protein 1 (PfEMP1). PfEMP1s are expressed on the surface of infected red blood cells (iRBCs) and play an essential role in sequestering the iRBC in tissue microvasculature, thus avoiding clearance in the spleen. The var genes that encode PfEMP1 are highly polymorphic and each parasite clone expresses one var at a time but can readily switch to the expression of an antigenically unrelated var under immune pressure. PfEMP1s can be classified into three groups, A, B and C. Current evidence suggest that antibodies to Var A are acquired first in children in endemic areas and that var As are involved in the most severe forms of malaria. Thus, understanding the acquisition of PfEMP1s may aid in the development of vaccines that prevent severe malaria. We will screen serum from our cohort for antibodies to the var A, B and C PfEMP1s using proteins produced by the Copenhagen group and establish the order of acquisition of specific antibodies and their longevity. Although long-lived classical MBCs (CD19+/CD20+/CD21+/CD27+/CD10-) are gradually acquired in response to natural infection, exposure to P. falciparum also results in a large expansion of what we have termed atypical MBCs (CD19+/CD20+/CD21-/CD27-/CD10-). At present, the function of atypical MBCs in malaria is not known nor are the factors that drive their differentiation. To gain insight into the relationship between classical and atypical IgG+ MBCs we compared the Ab heavy and light chain V gene repertoires of children living in a malaria endemic region in Mali. We found that these repertoires were remarkably similar by a variety of criteria including V gene usage, rate of somatic hypermutation and CDR-H3 length and composition. The similarity in these repertoires suggests that classical MBCs and atypical MBCs differentiate in response to similar Ag-dependent selective pressures in malaria exposed children and that atypical MBCs do not express a unique V gene repertoire. We have recently expanded our analysis of the antibody repertoire to describe the structure of the entire human antibody repertoire in response to malaria in collaboration with Dr. Ning Jiang, University of Texas. To do so we will use high-throughput-long read sequencing to characterize the expressed antibody repertoire in children and adults living in malaria-endemic Mali and the impact of malaria on this repertoire. We will also clone VH and VL from individual B cells and express those as antibodies to determine which sequences contribute to the Pf-specific immune response. Over the last year we also explored the relationship between Pf infections and autoimmune disease. These studies are based on a long standing observation of a link between autoimmune disease and malaria, namely that in malaria endemic regions of Africa there is very little autoimmune disease. This observation suggests that susceptibility to autoimmune disease may be modulated by malaria and/or that susceptibility to autoimmune disease is protective against sever malaria. In collaboration with Dr. Silvia Bolland, we provided evidence that a genetic susceptibility to systemic lupus erythematosus (SLE) protects against cerebral malaria in a mouse model. Protection appears to be by immune mechanisms that allow SLE-prone mice to better control their overall inflammatory responses to parasite infections. We are exploring the possibility that anti-inflammatory therapy may treat cerebral malaria. In addition, in collaboration with Dr. Inaki Sanz (Emory University) we are characterizing our Malian cohort for autoantibodies associated with human SLE. VH4-34-containing antibodies are auto-antibodies and can be identified using the VH4-34-specific monoclonal antibody 9G4. 9G4+ memory B cells and serum antibodies are prevalent in SLE but not in healthy controls. We have recently determined that in malaria-exposed Malians 9G4+ antibodies rise after clinical malaria but are controlled and rapidly disappear and do not appear in the memory responses. These observations suggest a special but distinct regulation of 9G4+ B cells and antibodies in SLE and in malaria. To better characterize the sequence of events that contribute to cerebral malaria we have established a collaboration with Dr. Dorian McGavern to use two photon, intravital imaging to view the cellular events that occur following infection with a mouse Plasmodium that results in cerebral disease using fluorescently labeled parasites, T cell, mononuclear cells and markers for vascular permeability. These studies have provided strong evidence that the interaction of parasite-specific CD8+ T cells with brain endothelium is necessary for the pathology of cerebral malaria. These results have led us to test two inhibitors of T cell metabolism as adjunctive therapy for cerebral malaria. Our results thus far are promising showing that one inhibitor of glutamine biosynthesis given to clinically ill infected mice reverses the brain damage and promotes healing.